Seven-15% of women who undergo breast conservation surgery have local recurrences, despite seemingly adequate surgical margins, consolidation with radiation therapy and adjuvant systemic therapies1. This is thought to be caused by residual cancer cells outside the surgical margin2-4. In contrast, we have shown that the cells in histologically normal tissue up to 1 cm away from breast tumors have several molecular alterations that suggest they may also contribute to local recurrence. The epithelial populations demonstrate genomic instability5, and a mechanism for immortalization6-7 (hTERT expression levels similar to those found in tumors). Furthermore, the fibroblasts within these tissues exhibit wound healing properties, which is a characteristic of Cancer Associated Fibroblasts8 (CAFs) and known to promote tumorigenesis9. We term tumor adjacent tissue with these abnormal characteristics Field Cancerized Tissue (FCT). Since an adequate surgical margin for a lumpectomy is defined as 2 mm, and FCT tissue extends at least 1 cm from the margin, FCT often remains in a woman after breast conserving surgery. However, it is not known if the cells within this tissue are pre- disposed to tumorigenesis, and thus contribute to local recurrence. Our long-term goal is to understand if the molecular alterations in FCT are a cause of local recurrence. The objective of this proposal is to understand the tumorigenic potential of cells within FCT in vitro. Our central hypothesis is that the molecular abnormalities n both epithelial and fibroblast cells from FCT confer tumorigenic properties. Aim 1 will determine if epithelial cells from FCT exhibit tumorigenic properties known to be conferred by hTERT. hTERT confers mitogen independence and immortalize cells and, in combination with other oncoproteins, tumorigenically transforms cells. We hypothesize that TAHN-1 epithelial cells also share these properties. Aim 2 will compare SDF-1 and TGF- signaling between fibroblasts in FCT and CAFs. CAFs secrete SDF-1 and TGF, which function to auto-stimulate their own proliferation along with that of adjacent epithelial cells. We predict that TAHN-1 fibroblasts also secrete these cytokines, and that their observed autostimulatory growth is dependent on these cytokines. Aim 3 will assess the tumorigenic potential of co-mixed epithelial cells and fibroblasts from FCT in vitro. CAFs cause the progression of initiated (i.e., expressing SV40 Large T- antigen) non-tumorigenic epithelial cells in vitro and in vivo9; however, CAFs do not cause progression of non-initiated cells9. We anticipate that CAFs will stimulate progression of TAHN-1 epithelial cells; and that TAHN-1 fibroblasts will stimulate progression of initiated and TAHN-1 epithelial cells. We will also test if TAHN-1 fibroblasts can stimulate progression of TAHN-1 epithelial cells. The proposed investigations will provide the first evaluation of the tumorigenic potential of the histologically normal tissue that often remains after breast conserving surgery. This proposal challenges the assumption that local recurrence is always due to residual cancer cells and, therefore, has implications on the adequacy of current standards for surgical margins in breast cancer. PUBLIC HEALTH RELEVANCE: We have shown that the histologically normal tissue up to 1 cm away from breast tumors has molecular changes that may pre-dispose this tissue for tumor formation. This relates to public health because adequate surgical margins can be as little as 2 mm, therefore this tissue likely remains in many women following breast conserving surgery. Our long-term goal is to understand if the molecular alterations in this tissue are a cause of loca recurrence. The objective of this proposal is to understand the potential of cells within this tisse to become cancer cells in tissue culture.